Gene concerning brassinosteroid-sensitivity of plants and utilization thereof

ABSTRACT

The present inventors successfully produced rice dwarf mutant d61 and also isolated the OsBRI1 gene which corresponds to a region in the d61 locus. OsBRI1 is found to increase plant brassinosteroid sensitivity. Moreover, the present inventors showed that OsBRI1 functions in growth and development process of rice, such as, internode elongation by inducing internode cell elongation and the inclination of the lamina joint. By introducing antisense nucleotides or dominant negative of OsBRI1, the present inventors produced transgenic rice plants whose phenotype was modified.

This application is a National Stage Application of InternationalApplication Number PCT/JP01/02770, published, pursuant to PCT Article21(2).

TECHNICAL FIELD

The present invention relates to a novel gene involved in plantbrassinosteroid sensitivity, the protein encoded by the gene, andproduction and use of the same.

BACKGROUND ART

Research in plant molecular biology has advanced dramatically in recentyears and is necessary for the analysis of various physiologicalphenomena. Dwarfism caused by artificial modification of grass type,especially the control of elongation growth, prevents plants fromlodging due to overgrowth caused by over fertilization. This preventionof lodging was demonstrated in Mexican wheat during the “GreenRevolution” and in miracle rice (IR-8) developed by the InternationalRice Research Center. Furthermore, in the case of cultivation at highdensity, such as rice cultivation, yields are expected to increase as aresult of the increase in the amount of sun light each plant receivesdue to the formation of upright leaves. Moreover, these modificationsare very important breeding targets because they may result in yieldincreases and also increase the efficiency of plant growth maintenance.However, current breeding methods cannot artificially modify plantmorphology.

Dwarfism is an abnormal growth caused by mutation in genes involved incontrolling normal elongation growth. Plant elongation growth is theresult of accumulation of cell division and cell elongation. Celldivision and cell elongation are controlled by complex effects caused byvarious factors, such as, exogenous environmental factors includingtemperature and light and endogenous environmental factors includingplant hormones. Therefore, it is predicted that many genes, such asthose related to plant hormone biosynthesis and hormone receptorsdirectly and those related to the control of the expression of thesegenes, are involved in the dwarfism (Sakamoto et al. (2000) Kagaku toSeibutsu, 38: 131–139).

Almost all modern cultivars of japonica rice develop 15–16 phytomers,consisting of leaves, axillary buds, and short or elongated internodes,during the vegetative stage. After the shoot meristem shifts from thevegetative to the reproductive phase, the reproductive meristem developsabout 10 phytomers consisting of undeveloped leaf, an elongatedinternode, and an axillary which develops into the primary rachisbranch. The phytomers formed in the vegetative stage can be classifiedinto three types in terms of the morphology of the internode (Suetsugu,Isao. (1968) Japan. J. Crop Sci. 37, 489–498). The first type isdeveloped in the juvenile phase and form undifferentiated nodes andinternodes. After the shoot apical meristem (SAM) shifts from thejuvenile to the adult phase, the nodal plate of the second typedifferentiates and the central part of the internode thereof decays toproduce an air space. The third type contains long elongated internodesas a result of growth from the intercalary meristem.

Phytomers of type 1 are produced first during vegetative development,followed by type 2 and then type 3 phytomers. Under normal growthconditions, the number of phytomers of each type in many japonicacultivars is 4–5, 6–7, and 4–5. The transition from type 1 to type 2 isstrictly regulated. After the serial development of 4–5 type 1phytomers, depending on the cultivar, the SAM develops type 2 phytomers.However, the transition from type 2 to type 3 does not depend on thenumber of development of type 2 phytomers. The SAM develops 15–16phytomers and shifts from the vegetative to the reproductive stage, thetype 3 phytomers then start to develop, and the uppermost four or fiveinternodes thereof start to elongate. If the timing of the transition ischanged by unusual growth conditions, the number of the type 2 phytomersalways affected thereby, but the number of the type 3 phytomer withelongated internode is unchanged (Suetsugu, isao. (1968) Japan. J. CropSci: 37, 489–498). This indicates that the transition of the SAM fromthe vegetative to the reproductive phase in rice induces internodeelongation, as well as in Arabidopsis.

However, there is an important difference between rice and Arabidopsis.The elongated internodes in rice are derived from the vegetative SAMwhile those in Arabidopsis come from the reproductive SAM. In rice, theuppermost four or five internodes develop from the vegetative SAM andinitially are indistinguishable from the lower type 2 internodes. Whenthe SAM shifts to the reproductive phase, differentiation into type 3internodes occurs due to the development of intercalary meristems in theinternodes. This synchronicity between the phase change of the SAM andthe development of the intercalary meristem leads to the possibilitythat these processes might be linked by a signal coming from the SAM tothe uppermost four or five phytomers when its phase change occurs.

A large number of dwarf mutants of rice have been collected andcharacterized because of their agronomic importance. These dwarf mutantsare categorized into six groups based on the elongation pattern of theupper four to five internodes (FIG. 1; redrawn from Takeda, K. (1974)Bull. Fac. Agr. Hirosaki Univ. 22, 19–30. In rice, each internode isnumbered from top to bottom such that the uppermost internode just belowthe panicle is first). The present inventors can see that in the dn-typemutants the length of each internode is almost uniformly reduced,resulting in an elongation pattern similar to that of the wild typeplant. In contrast, the dm-type mutants show specific reduction of thesecond internode. Similar shortening of a specific internode is alsoobserved in the sh- and d6-type mutants, in which only the uppermostfirst internode or internodes below the uppermost are shortened,respectively. As these mutants with specifically shortened internodes,such as the dm-, d6-, and sh-types, might be defective in the perceptionof signals coming from the SAM, they should be especially useful for thestudy of the mechanism of internode elongation and its relationship tochanges in the SAM.

Brassinosteroids (BRs) are plant growth-promoting natural products thatare required for plant growth and development. There are only a fewreports on the physiological effects of brassinosteroids in the growthand development of rice and other plants of the Gramineae family.Physiological researches indicate that exogenous brassinosteroids alone,or in combination with auxin, enhance bending of the lamina joint inrice. The lamina joint has been used for a sensitive bioassay ofbrassinosteroids (Maeda, E. (1965) Physiol. Plant. 18, 813–827; Wada, K.et al. (1981) Plant and Cell Physiol. 22, 323–325; Takeno, K. andPharis, R. P. (1982) Plant Cell Physiol. 23, 1275–1281), because of highsensitivity thereof to brassinosteroids. In etiolated wheat seedlingstreatment with brassinolide or its derivative, castasterone, stimulatesunrolling of the leaf blades (Wada, K. et al. (1985) Agric. Biol. Chem.49, 2249–2251). Treatment with low or high concentrations ofbrassinosteroids promotes or inhibits the growth of roots in rice,respectively (Radi, S. H. and Maeda, E. (1988) J. Crop Sci. 57,191–198). Brassinosteroids also promote the germination of rice seeds(Yamaguchi, T. et al. (1987) Stimulation of germination in aged riceseeds by pre-treatment with brassinolide. In: Proceeding of thefourteenth annual plant growth regulator society of America MeetingHonolulu. (Cooke A R), pp. 26–27).

Although these results indicate only effects due to exogenousbrassinosteroids, not due to endogenous brassinosteroids, they dosuggest that endogenous brassinosteroids have an important role ingrowth and developmental processes in plants of the Gramineae family.

On the other hand, there is some apparent disagreement in the literatureas to whether brassinosteroids induce cell elongation in plants of theGramineae family. That is, brassinolide treatment does not induceelongation of the leaf sheath of rice (Yokota, T. and Takahashi, N.(1986) Chemistry, physiology and agricultural application ofbrassinolide and related steroids. In: Plant growth substances 1985.(Bopp M, Springer-Verlag, Berlin/Heidelberg/New York) pp.129–138), butit does induce elongation of the coleoptile and mesocotyl in maize (He,R. —Y. et al. (1991) Effects of brassinolide on growth and chillingresistance of maize seedlings. In: Brassinosteroids-Chemistry,Bioactivity and Applications ACS symposium series 474. (Cutler H G C,Yokota T, Adam G, American Chemical Society, Washington D.C.), pp.220–230).

As shown by brassinosteroids synthesis mutants or brassinosteroidsinsensitive mutants that show severe dwarfism with abnormal developmentof organs, the function of brassinolide is known in dicotyledonousplants.

However, little is known about the function of endogenousbrassinosteroids in monocotyledonous plants, such as rice or otherplants of the Gramineae family.

3. Disclosure of the Invention

The object of the present invention is to provide novel genes involvedin brassinosteroid sensitivity from plants, preferably frommonocotyledonous plants. Another object of the present invention is tomodify plant brassinosteroid sensitivity by controlling the expressionof the gene. The modification in plant brassinosteroid sensitivitycauses a change in plant morphology. The preferable embodiment of thepresent invention provides plants with erect leaves which become dwarfeddue to the suppression of internode elongation caused by decreasedbrassinosteroid sensitivity.

By treatment with mutagenesis agent, the present inventors isolated anovel rice dwarf mutant strain d61 (d61-1 and d61-2) which showed lowerbrassinosteroid sensitivity and had shorter internodes than wild typeplants.

Linkage analysis indicated that the d61 locus was highly linked to agene region that was homologous to Arabidopsis BRI1. The presentinventors isolated the gene (OsBRI1), which was homologous toArabidopsis BRI1 gene, by screening of a rice genomic DNA library.Nucleotide sequence analysis of the OsBRI1 gene from d61-1 and d61-2mutants indicated that there were single nucleotide substitutionscausing amino acid substitutions at different sites in each d61 allele.

Moreover, in order to confirm that the OsBRI1 gene corresponds to thed61 locus, the OsBRI1 gene was introduced into d61 mutants. As a result,the OsBRI1 gene complimented the d61 phenotype and caused the mutantstrain to have a wild-type phenotype. Therefore, it was indicated thatd61 mutants are caused by loss of function of the OsBRI1 gene.Phenotypic analysis of plants revealed that the OsBRI1 gene functions invarious growth and development processes of rice including internodeelongation caused by formation of intercalary meristem and induction ofinternode cell longitudinal elongation, inclination of the lamina joint,and skotomorphogenesis in the dark.

Moreover, in the case where transgenic rice plants with OsBRI1 antisensenucleotide were produced, most transgenic plants produced erect leavesduring seedling growth. All of the transgenic plants showed dwarfphenotype of various levels. Plants transformed with OSBRI1 having thedominant negative phenotype showed the same result.

The present invention had been made in view of such findings, andrelates to a novel gene involved in plant brassinosteroid sensitivity,the protein encoded by the gene, and production and use of the same.Moreover, the present invention relates to the production of modifiedplant by controlling expression of the gene.

More specifically, this invention provides:

-   (1) a DNA encoding a protein comprising the amino acid sequence of    SEQ ID NO: 2;-   (2) the DNA of (1), wherein the DNA is a cDNA or a genomic DNA;-   (3) the DNA of (1), wherein the DNA comprises a coding region of the    nucleotide sequence of SEQ ID NO: 1 or 3;-   (4) a DNA encoding a protein which has 55% or more homology to the    amino acid sequence of SEQ ID NO: 2 and which is functionally    equivalent to a protein comprising the amino acid sequence of SEQ ID    NO: 2, the DNA being selected from the group consisting of    -   (a) a DNA encoding a protein comprising the amino acid sequence        of SEQ ID NO: 2 in which one or more amino acids are        substituted, deleted, added, and/or inserted; and    -   (b) a DNA hybridizing under stringent conditions with a DNA        comprising the nucleotide sequence of SEQ ID NO: 1 or 3;-   (5) the DNA of (4), wherein the DNA encodes a protein having a    function selected from the group consisting of a function of    increasing brassinosteroid sensitivity in a plant, a function of    inducing elongation of internode cells of a stem of a plant, a    function of positioning microtubules perpendicular to the direction    of elongation in an internode of a stem of a plant, a function of    suppressing elongation of an internode of a neck of a plant, and a    function of increasing inclination of a lamina of a plant;-   (6) the DNA of (4) or (5), wherein the DNA is derived from a    monocotyledonous plant;-   (7) the DNA of (6), wherein the DNA is derived from a plant of the    Gramineae family;-   (8) a DNA encoding an antisense RNA complementary to a transcript of    the DNA of any one of (1) to (7);-   (9) a DNA encoding an RNA having ribozyme activity which    specifically cleaves a transcript of the DNA of any one of (1) to    (7);-   (10) a DNA which encodes an RNA repressing expression of the DNA of    any one of (1) to (7) due to co-suppression when expressed in a    plant cell and which has 90% or more homology to the DNA of any one    of (1) to (7);-   (11) a DNA which encodes a protein having a dominant negative    phenotype to that of a protein encoded by the DNA of any one of (1)    to (7);-   (12) a vector which comprises the DNA of any one of (1) to (7);-   (13) a transformed cell which comprises the DNA of any one of (1)    to (7) or the vector of (12);-   (14) a protein encoded by the DNA of any one of (1) to (7);-   (15) a method for producing the protein of (14) the method    comprising the steps of culturing the transformed cell of (13) and    recovering an expressed protein from the transformed cell or a    culture supernatant thereof;-   (16) a vector comprising the DNA of any one of (8) to (11);-   (17) a transformed plant cell comprising the DNA of any one of (1)    to (11) or the vector of (12) or (16);-   (18) a transformed plant comprising the transformed plant cell of    (17);-   (19) a transformed plant which is a progeny or a clone of the    transformed plant of (18);-   (20) a breeding material of the transformed plant of (18) or (19);    and-   (21) an antibody which binds to the protein of (14).

The present invention provides a DNA encoding the OsBRI1 protein derivedfrom rice. The nucleotide sequence of OsBRI1 cDNA is shown in SEQ ID NO:1, the amino acid sequence of the protein encoded by the DNA is shown inSEQ ID NO: 2, and the nucleotide sequence of the genomic DNA of OsBRI1is shown in SEQ ID NO: 3 (the genomic DNA of SEQ ID NO: 3 consists ofone exon with no intron).

The gene of the present invention causes a rice dwarf mutant (d61) whichhas short internodes and reduced brassinosteroid sensitivity compared tothe wild type. Therefore, it is possible to modify plant morphology bycontrolling the expression of the OsBRI1 gene.

The preferable modification in plant morphology in the present inventionincludes dwarfism of plants by suppressing expression of the DNA of thepresent invention. Dwarfism of plants has great value in agriculture andhorticulture. For example, reduction of height of plants can reduce thetendency of plants to lodge and can thereby increase seed weights.Moreover, it is possible to increase the number of plant individualswhich can be planted per unit area by reducing height of plants and bymaking plant shape per plant more compact. These plant modificationshave great value specifically in the production of crops such as rice,corn, wheat, and such. It is also possible to produce ornamental plantswith new aesthetic value by dwarfism of height or culm length of plants.It is also possible to produce miniature vegetables or fruits with newcommercial value, such as “bite-size”, by dwarfism of them. Other thanfor industrial plants, dwarfism is important for experimental plantsbecause, for example, dwarf plants are not only more easily handled butthey also help utilize experimental space more effectively by decreasingcultivation space.

It is possible to consider that brassinosteroid sensitivity can beincreased in brassinosteroid low sensitive plants by expressing the DNAof the present invention in the plants. Thereby, the yield of wholeplants may be increased by growing taller plants. Thus, this will beespecially useful for increasing yield for whole feed crops.

DNA encoding the OsBRI1 protein of the present invention includesgenomic DNA, cDNA, and chemically synthesized DNA. A genomic DNA andcDNA can be prepared according to conventional methods known to thoseskilled in the art. More specifically, a genomic DNA can be prepared,for example, as follows: (1) extract genomic DNA from plant cells ortissues; (2) construct a genomic library (utilizing a vector, such asplasmid, phage, cosmid, BAC, PAC, and such); (3) spread the library; and(4) conduct colony hybridization or plaque hybridization using a probeprepared based on the DNA encoding a protein of the present invention(e.g., SEQ ID NO: 1 or 3). Alternatively, a genomic DNA can be preparedby PCR, using primers specific to a DNA encoding the protein of thepresent invention (e.g. SEQ ID NO: 1 or 3). On the other hand, cDNA canbe prepared, for example, as follows: (1) synthesize cDNAs based onmRNAs extracted from plant cells or tissues; (2) prepare a cDNA libraryby inserting the synthesized cDNA into vectors, such as λZAP; (3) spreadthe cDNA library; and (4) conduct colony hybridization or plaquehybridization as described above. Alternatively, cDNA can be alsoprepared by PCR.

The present invention includes DNAs encoding proteins functionallyequivalent to the OsBRI1 protein of SEQ ID NO: 2. Herein, the term“functionally equivalent to the OsBRI1 protein” means that the objectprotein has equal functions to those of the OsBRI1 protein of SEQ ID NO:2, such as, for example, a function of increasing brassinosteroidsensitivity in a plant, a function of inducing elongation of aninternode of a stem of a plant, a function of positioning microtubulesperpendicular to the direction of elongation in internode cells of astem of a plant, a function of suppressing elongation of an internode ofa neck of a plant, and/or a function of increasing inclination of alamina of a plant. Such DNA is derived preferably from monocotyledonousplants, more preferably from plants of the Gramineae family, and mostpreferably from rice.

Examples of such DNAs include those encoding mutants, derivatives,alleles, variants, and homologues comprising the amino acid sequence ofSEQ ID NO: 2 wherein one or more amino acids are substituted, deleted,added, and/or inserted.

Examples of methods for preparing a DNA encoding a protein comprisingaltered amino acids well known to those skilled in the art include thesite-directed mutagenesis (Kramer, W. and Fritz, H. -J. (1987)“Oligonucleotide-directed construction of mutagenesis via gapped duplexDNA.” Methods in Enzymology, 154: 350–367). The amino acid sequence of aprotein may also be mutated in nature due to the mutation of anucleotide sequence. A DNA encoding proteins having the amino acidsequence of a natural OsBRI1 protein (SEQ ID NO: 2) wherein one or moreamino acids are substituted, deleted, and/or added are also included inthe DNA of the present invention, so long as they encode a proteinfunctionally equivalent to the natural OsBRI1 protein. Additionally,nucleotide sequence mutants that do not give rise to amino acid sequencechanges in the protein (degeneracy mutants) are also included in the DNAof the present invention. The number of nucleotide mutations of the DNAof interest corresponds to, at amino acid level, typically 100 residuesor less, preferably 50 residues or less, more preferably 20 residues orless, and still more preferably 10 residues or less (for example, 5residues or less, or 3 residues or less).

Whether a certain DNA actually encodes a protein which has a function ofincreasing inclination of a lamina of a plant can be evaluated, forexample, by performing a “lamina joint test” for plants in which theexpression of the DNA has been suppressed and by comparing the resultswith those for wild-type plants (See Example 4). The result of the testmay also be an index for evaluating brassinosteroid sensitivity in aplant. In order to evaluate whether the DNA encodes a protein which hasa function of inducing elongation of an internode of a stem of a plant,a function of positioning microtubules perpendicular to the direction ofelongation in internode cells of a stem of a plant, or a function ofsuppressing elongation of an internode of a neck of a plant, themorphology of the internode cell of the plant in which expression of theDNA has been suppressed can be observed to be compared with that of wildtype (See Examples 2 and 3).

A DNA encoding a protein functionally equivalent to the OsBRI1 proteindescribed in SEQ ID NO: 2 can be produced, for example, by methods wellknown to those skilled in the art including: methods using hybridizationtechniques (Southern, E. M. (1975) Journal of Molecular Biology, 98,503); and polymerase chain reaction (PCR) techniques (Saiki, R. K. etal. (1985) Science, 230, 1350–1354; Saiki, R. K. et al. (1988) Science,239, 487–491). That is, it is routine for a person skilled in the art toisolate a DNA with high homology to the OsBRI1 gene from rice and otherplants using the OsBRI1 gene (SEQ ID NO: 1 or 3) or parts thereof as aprobe, and oligonucleotides hybridizing specifically to the gene as aprimer. Such DNA encoding proteins functionally equivalent to the OsBRI1protein, obtainable by hybridization techniques or PCR techniques, areincluded in the DNA of this invention.

Hybridization reactions to isolate such DNAs are preferably conductedunder stringent conditions. Stringent hybridization conditions of thepresent invention include conditions such as: 6 M urea, 0.4% SDS, and0.5×SSC; and those which yield a similar stringency with the conditions.DNAs with higher homology are expected to be isolated efficiently whenhybridization is performed under conditions with higher stringency, forexample, 6 M urea, 0.4% SDS, and 0.1×SSC. Those DNAs isolated under suchconditions are expected to encode a protein having a high amino acidlevel homology with OsBRI1 protein (SEQ ID NO: 2). Herein, “highhomology” means an identity of at least 55% or more, more preferably 70%or more, and most preferably 90% or more (e.g., 95% or more), betweenfull-length of amino acids.

The degree of homology of one amino acid sequence or nucleotide sequenceto another can be determined by following the algorithm BLAST by Karlinand Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873–5877, 1993). Programssuch as BLASTN and BLASTX were developed based on this algorithm(Altschul et al. J. Mol. Biol. 215: 403–410, 1990). To analyze anucleotide sequences according to BLASTN based on BLAST, the parametersare set, for example, as score=100 and word length=12. On the otherhand, parameters used for the analysis of amino acid sequences by theBLASTX based on BLAST include, for example, score=50 and word length=3.Default parameters of each program are used when using BLAST and GappedBLAST program. Specific techniques for such analysis are known in theart.

The DNA of the present invention can be used, for example, to preparerecombinant proteins, produce transformed plants with phenotypes alteredby controlling expression thereof as described above, and so on.

A recombinant protein is usually prepared by inserting a DNA encoding aprotein of the present invention into an appropriate expression vector,introducing said vector into an appropriate cell, culturing thetransformed cells, and purifying expressed proteins.

A recombinant protein can be expressed as a fusion protein with otherproteins so as to be easily purified, for example, as a fusion proteinwith maltose binding protein in Escherichia coli (New England Biolabs,USA, vector pMAL series), as a fusion protein withglutathione-S-transferase (GST) (Amersham Pharmacia Biotech, vector pGEXseries), or tagged with histidine (Novagen, pET series). The host cellis not limited so long as the cell is suitable for expressing therecombinant protein. It is possible to utilize yeasts or various animal,plant, or insect cells besides the above described E. coli. A vector canbe introduced into a host cell by a variety of methods known to oneskilled in the art. For example, a transformation method using calciumions (Mandel, M. and Higa, A. (1970) Journal of Molecular Biology, 53,158–162; Hanahan, D. (1983) Journal of Molecular Biology, 166, 557–580)can be used to introduce a vector into E. coli. A recombinant proteinexpressed in host cells can be purified and recovered from the hostcells or the culture supernatant thereof by known methods. When arecombinant protein is expressed as a fusion protein with maltosebinding protein or other partners, the recombinant protein can be easilypurified by affinity chromatography.

The resulting protein can be used to prepare an antibody that binds tothe protein. For example, a polyclonal antibody can be prepared byimmunizing immune animals, such as rabbits, with a purified protein ofthe present invention or its portion, collecting blood after a certainperiod, and removing clots. A monoclonal antibody can be prepared byfusing myeloma cells with the antibody-forming cells of animalsimmunized with the above protein or its portion, isolating a monoclonalcell expressing a desired antibody (hybridoma), and recovering theantibody from the cell. The obtained antibody can be utilized to purifyor detect a protein of the present invention. Accordingly, the presentinvention includes antibodies that bind to proteins of the invention.

In order to produce a transformed plant in which DNAs of the presentinvention are expressed, a DNA encoding a protein of the presentinvention is inserted into an appropriate vector; the vector is thenintroduced into a plant cell; and finally, the resulting transformedplant cell is regenerated.

On the other hand, a transformed plant with suppressed expression ofDNAs of the present invention can be created using DNA that repressesthe expression of a DNA encoding a protein of the present invention:wherein the DNA is inserted into an appropriate vector, the vector isintroduced into a plant cell, and then, the resulting transformed plantcell is regenerated. The phrase “suppression of expression of DNAencoding a protein of the present invention” includes suppression ofgene transcription as well as suppression of translation into protein.It also includes not only the complete inability of expression of DNAbut also reduction of expression.

The expression of a specific endogenous gene in plants can be repressedby methods utilizing antisense technology, the methods which arecommonly used in the art. Ecker et al. were the first to demonstrate theantisense effect of an antisense RNA introduced by electroporation inplant cells by using the transient gene expression method (J. R. Eckerand R. W. Davis (1986) Proc. Natl. Acad. Sci. USA 83: 5372). Thereafter,the target gene expression was reportedly reduced in tobacco andpetunias by expressing antisense RNAs (A. R. van der Krol et al. (1988)Nature 333: 866). The antisense technique has now been established as ameans to repress target gene expression in plants.

Multiple factors are required for antisense nucleic acid to repress thetarget gene expression. These include, inhibition of transcriptioninitiation by triple strand formation; suppression of transcription byhybrid formation at the site where the RNA polymerase has formed a localopen loop structure; transcription inhibition by hybrid formation withthe RNA being synthesized; suppression of splicing by hybrid formationat the junction between an intron and an exon; suppression of splicingby hybrid formation at the site of spliceosome formation; suppression ofmRNA translocation from the nucleus to the cytoplasm by hybrid formationwith mRNA; suppression of splicing by hybrid formation at the cappingsite or at the poly A addition site; suppression of translationinitiation by hybrid formation at the binding site for the translationinitiation factors; suppression of translation by hybrid formation atthe site for ribosome binding near the initiation codon; inhibition ofpeptide chain elongation by hybrid formation in the translated region orat the polysome binding sites of mRNA; and suppression of geneexpression by hybrid formation at the sites of interaction betweennucleic acids and proteins. These factors repress the target geneexpression by inhibiting the process of transcription, splicing, ortranslation (Hirashima and Inoue, “Shin Seikagaku Jikken Koza (NewBiochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV,Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes)”,Nihon Seikagakukai Hen (The Japanese Biochemical Society), Tokyo KagakuDozin, pp. 319–347, (1993)).

An antisense sequence of the present invention can repress the targetgene expression by any of the above mechanisms. In one embodiment, if anantisense sequence is designed to be complementary to the untranslatedregion near the 5′ end of the gene's mRNA, it will effectively inhibittranslation of a gene. It is also possible to use sequencescomplementary to the coding regions or to the untranslated region on the3′ side. Thus, the antisense DNA used in the present invention includesDNA having antisense sequences against both the untranslated regions andthe translated regions of the gene. The antisense DNA to be used isconnected downstream from an appropriate promoter, and, preferably, asequence containing the transcription termination signal is connected onthe 3′ side. The DNA thus prepared can be transfected into the desiredplant by known methods. The sequence of the antisense DNA is preferablya sequence complementary to the endogenous gene of the plant to betransformed or a part thereof, but it need not be perfectlycomplementary so long as it can effectively inhibit the gene expression.The transcribed RNA is preferably at least 90%, and most preferably atleast 95% complementary to the transcribed products of the target gene.Sequence complementarity may be determined using the above-describedsearch.

In order to effectively inhibit the expression of the target gene bymeans of an antisense sequence, the antisense DNA should be at least 15nucleotides long, preferably at least 100 nucleotides long, and morepreferably at least 500 nucleotides long. The antisense DNA to be usedis generally shorter than 5 kb, and preferably shorter than 2.5 kb.

DNA encoding ribozymes can also be used to repress the expression ofendogenous genes. A ribozyme is an RNA molecule that has catalyticactivity. There are many ribozymes having various activities. Researchon ribozymes as RNA cleaving enzymes has enabled the design of aribozyme that site-specifically cleaves RNA. While some ribozymes of thegroup I intron type or the mRNA contained in RNaseP consist of 400nucleotides or more, others belonging to the hammerhead type or thehairpin type have an activity domain of about 40 nucleotides (MakotoKoizumi and Eiko Ohtsuka, (1990) Tanpakushitsu Kakusan Kohso (Nucleicacid, Protein, and Enzyme), 35: 2191).

The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′side of C15 of the sequence G13U14C15. Formation of a nucleotide pairbetween U14 and A at the ninth position is considered important for theribozyme activity. Furthermore, it has been shown that the cleavage alsooccurs when the nucleotide at the 15th position is A or U instead of C(M.. Koizumi et al., (1988) FEBS Lett. 228: 225). If the substratebinding site of the ribozyme is designed to be complementary to the RNAsequences adjacent to the target site, one can create arestriction-enzyme-like RNA cleaving ribozyme which recognizes thesequence UC, UU, or UA within the target RNA (M. Koizumi et al., (1988)FEBS Lett. 239: 285; Makoto Koizumi and Eiko Ohtsuka, (1990)Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35:2191; M. Koizumi et al., (1989) Nucleic Acids Res. 17: 7059). Forexample, in the coding region of the OsBRI1 gene (SEQ ID NO: 1 or 3),there is a plurality of sites that can be used as the ribozyme target.

The hairpin type ribozyme is also useful in the present invention. Ahairpin type ribozyme can be found, for example, in the minus strand ofthe satellite RNA of tobacco ringspot virus (J. M. Buzayan, Nature 323:349 (1986)). This ribozyme has also been shown to target-specificallycleave RNA (Y. Kikuchi and N. Sasaki, (1992) Nucleic Acids Res. 19:6751; Yo Kikuchi, (1992) Kagaku To Seibutsu (Chemistry and Biology) 30:112).

The ribozyme designed to cleave the target is fused with a promoter,such as the cauliflower mosaic virus 35S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. However, if extra sequences have been added to the 5′ endor the 3′ end of the transcribed RNA, the ribozyme activity can be lost.In this case, one can place an additional trimming ribozyme, whichfunctions in cis to perform the trimming on the 5′ or the 3′ side of theribozyme portion, in order to precisely cut the ribozyme portion fromthe transcribed RNA containing the ribozyme (K. Taira et al. (1990)Protein Eng. 3: 733; A. M. Dzaianott and J. J. Bujarski (1989) Proc.Natl. Acad. Sci. USA 86: 4823; C. A. Grosshands and R. T. Cech (1991)Nucleic Acids Res. 19: 3875; K. Taira et al. (1991) Nucleic Acid Res.19: 5125). Multiple sites within the target gene can be cleaved byarranging these structural units in tandem to achieve greater effects(N. Yuyama et al. (1992) Biochem. Biophys. Res. Commun. 186: 1271). Byusing such ribozymes, it is possible to specifically cleave thetranscripts of the target gene in the present invention, therebyrepressing the expression of said gene.

Endogenous gene expression can also be repressed by co-suppressionthrough the transformation by DNA having a sequence identical or similarto the target gene sequence. “Co-suppression” refers to the phenomenonin which, when a gene having a sequence identical or similar to thetarget endogenous gene sequence is introduced into plants bytransformation, expression of both the introduced exogenous gene and thetarget endogenous gene becomes repressed. Although the detailedmechanism of co-suppression is unknown, it is frequently observed inplants (Curr. Biol. (1996) 7: R793 (1997), Curr. Biol. 6: 810). Forexample, if one wishes to obtain a plant body in which the OsBRI1 geneis co-repressed, the plant in question can be transformed with a vectorDNA designed so as to express the OsBRI1 gene or DNA having a similarsequence to select a plant having the OsBRI1 mutant character, e.g., aplant with suppressed internode elongation, among the resultant plants.The gene to be used for co-suppression does not need to be completelyidentical to the target gene, but it should have at least 70% or moresequence identity, preferably 80% or more sequence identity, and morepreferably 90% or more (e.g., 95% or more) sequence identity. Sequenceidentity may be determined by above-described search.

In addition, endogenous gene expression in the present invention canalso be repressed by transforming the plant with a gene having thedominant negative phenotype of the target gene. Herein, “a DNA encodingthe protein having the dominant negative phenotype” refers to a DNAencoding a protein which, when the DNA is expressed, can eliminate orreduce the activity of the protein encoded by the endogenous gene of thepresent invention inherent to the plant. Preferably, it is a DNAencoding the peptide (e.g., peptide which contains from 739 to 1035residues of amino acids of SEQ ID NO: 2 or peptides of another proteinequivalent to the peptide) which lacks the N-terminal region butcontains the kinase region of the protein of the present invention.Whether the DNA of interest has the function to eliminate or enhanceactivity of the endogenous gene of the present invention can bedetermined, as mentioned above, by whether the DNA of interesteliminates or reduces a function of increasing brassinosteroidsensitivity in a plant, a function of inducing elongation of aninternode of a stem of a plant, a function of positioning microtubulesperpendicular to the direction of elongation in internode cells of astem of a plant, a function of suppressing elongation of an internode ofa neck of a plant, and/or a function of increasing inclination of alamina of a plant.

Vectors used for the transformation of plant cells are not limited aslong as the vector can express inserted genes in plant cells. Forexample, vectors comprising promoters for constitutive gene expressionin plant cells (e.g., califlower mosaic virus 35S promoter); andpromoters inducible by exogenous stimuli can be used. The term “plantcell” used herein includes various forms of plant cells, such ascultured cell suspensions, protoplasts, leaf sections, and callus.

A vector can be introduced into plant cells by known methods, such asthe polyethylene glycol method, electroporation, Agrobacterium mediatedtransfer, and particle bombardment. Plants can be regenerated fromtransformed plant cells by known methods depending on the type of theplant cell (Toki et al., (1995) Plant Physiol. 100:1503–1507). Forexample, transformation and regeneration methods for rice plantsinclude: (1) introducing genes into protoplasts using polyethyleneglycol, and regenerating the plant body (suitable for indica ricecultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”, Potrykus Iand Spangenberg Eds., pp66–74); (2) introducing genes into protoplastsusing electric pulse, and regenerating the plant body (suitable forjaponica rice cultivars) (Toki et al (1992) Plant Physiol. 100,1503–1507); (3) introducing genes directly into cells by the particlebombardment, and regenerating the plant body (Christou et al. (1991)Bio/Technology, 9: 957–962); (4) introducing genes using Agrobacterium,and regenerating the plant body; and so on. These methods are alreadyestablished in the art and are widely used in the technical field of thepresent invention. Such methods can be suitably used for the presentinvention.

Once a transformed plant, wherein the DNA of the present invention isintroduced into the genome, is obtained, it is possible to gaindescendants from that plant body by sexual or vegetative propagation.Alternatively, plants can be mass-produced from breeding materials (forexample, seeds, fruits, ears, tubers, tubercles, tubs, callus,protoplast, etc.) obtained from the plant, as well as descendants orclones thereof. Plant cells transformed with the DNA of the presentinvention, plant bodies including these cells, descendants and clones ofthe plant, as well as breeding materials obtained from the plant, itsdescendant and clones, are all included in the present invention.

The plant of the present invention is preferably a monocotyledonousplant, more preferably a plant of the Gramineae family, and mostpreferably a rice. The phenotype of the plant of the present inventionis different from the wild type phenotype. The phenotypes changed in theplants developed by the present invention include brassinosteroidsensitivity of a plant, plant growth such as internode cell elongationof the plant stem and internode elongation of the ear, inclination ofleaves, and the positioning of microtubules perpendicular to thedirection of internode cell elongation in the plant stem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the internode elongation pattern ofwild type rice and various dwarf mutant and wild-type rice plants. Therelative lengths of the each internode to the stem are shown in theschematic diagram. Wild type is shown as N.

FIG. 2 represents photographs which show the phenotype of the d61mutants.

(A) Gross morphology. (Left) Wild type plant; (centre) d61-1 mutant(weak allele); (right) d61-2 mutant (strong allele).

(B) Elongation pattern of internodes. The wild type plant (left) showsthe N-type of the elongation pattern, while the d61-1 (centre) and d61-2(right) mutants show typical dn- and d6-type patterns, respectively.

(C) Panicle structure. The wild type plant (left) has a short panicle,while the d61-1 (centre) and d61-2 (right) mutants have longer panicles.

(D) Erect leaf of d61. The leaves of the wild type plant (left) are bentat the lamina joint indicated by the white arrow, while the leaves ofd61-1 (centre) and d61-2 (right) mutants are more erect.

(E) Leaf sheath of d61. The leaf sheath in the d61-1 (centre) and d61-2(right) mutants is shorter than in the wild type plants (left).

FIG. 3 represents microphotographs which show the structure ofwell-developed internodes from wild type and d61-2 rice plants, asfollows:

(A) longitudinal sections of the first internodes from wild type;

(B) longitudinal sections of the second internodes from wild type;

(C) longitudinal sections of the third internodes from wild type;

(D) longitudinal sections of the fourth internodes from wild type;

(E) longitudinal sections of the first internodes from d61-2 riceplants;

(F) longitudinal sections of the second internodes from d61-2 riceplants;

(G) longitudinal sections of the third internodes from d61-2 riceplants; and

(H) longitudinal sections of the fourth internodes from d61-2 riceplants.

Bar=100 μm, respectively.

FIG. 4 are photographs and drawings which show the orientation ofmicrotubules in elongating sells in the first internode of wild type andd61-2 plants, consisting of immunofluorescence images (A and B) orschematic presentation (C and D) of the microtubule arrangement ininternodal parenchyma cells of the first internode from wild type (A andC) and d61-2 (B and D) plants. Bars=50 μm.

FIG. 5 is a photograph which shows the response of seedlings of wildplant, D61-1, and d61-2 to brassinolide.

Seeds were germinated on agar plates in the presence or absence of 1 μMbrassinolide (BL). Seedlings were observed 1 day after germination. BLtreatment induced abnormal growth in wild plant, while mutant seedlingswere not affected thereto.

FIG. 6 is a photograph which shows effect of brassinolide on the degreeof inclination of etiolated leaf lamina in wild type, d61-1, and d61-2plants.

The highest response of the leaf from wild type (panel A) and reducedresponse in mutant plants d61-1 (panel B) and d61-2 (panel C) are shown.

FIG. 7 is a drawing which shows amounts of brassinosteroids in wild typeand d61-2 rice plants, and biosynthetic precursors thereof.

The amounts (ng/g fresh weight) of each compound in mutant (upper) andwild type (lower) plants are shown. ND indicates not detected.

FIG. 8 is a photograph which shows de-etiolation phenotype of the d61 inthe dark.

(A) Wild Type

Left: seedlings grown for two weeks in the dark

Right: seedlings grown for two weeks in the light

The internode elongation in wild type (A) and two gibberellin deficientrice mutants, d18 (D) and d35 (E), are indicated in the dark.

(B) d61-1 mutant

Left: seedlings grown for two weeks in the dark

Right: seedlings grown for two weeks in the light

The white arrows indicate internode elongations in right of each panel,the dark condition. No elongation was observed in the light (left ofeach panel).

(C) d61-2 mutant

Left: seedlings grown for two weeks in the dark

Right: seedlings grown for two weeks in the light

No internode elongation in d61 mutant, d61-1 (B) and d61-2 (C), isobserved even in the dark. The present inventors stripped the leafsheath of plants grown in the dark.

(D) d18 mutant

Left: seedlings grown for two weeks in the dark

Right: seedlings grown for two weeks in the light

(E) d35 mutant

Left: seedlings grown for two weeks in the dark

Right: seedlings grown for two weeks in the light

FIG. 9 provides a drawing and photograph that show the strong linkagebetween the d61 locus and OsBRI1.

(A) is a drawing that indicates map position of the d61 locus on thelong arm of chromosome 1.

(B) is a photograph that indicates the result of DNA hybridizationanalysis to test the linkage between the d61 locus and OsBRI1. The RFLPof OsBRI1 was observed between Japonica parent T65 (lane T, 12.5 kb),and the Indica parent Kasarath (lane K, 17.5 kb) when the genomic DNAswere digested with EcoRI. Plants with a normal phenotype (WT) wereheterozygous (12.5+17.5 k.b) or homozygous for the Indica allele (17.5kb), while the plants with the mutant phenotype (mutant) were alwayshomozygous for the Japonica allele (12.5 kb).

FIG. 10 shows a comparison of the deduced amino acid sequences of OsBRI1(SEQ ID NO: 2 and Arabidopsis BRI1 (SEQ ID NO: 6). Identical residuesare boxed. The underlined regions, * 1, *2, *3, and *4, indicate: aputative signal peptide, a leucine zipper motif, N, and C sides of acysteine pair.

FIG. 11 is a continuation of FIG. 10.

FIG. 12 is a photograph which shows expression pattern of OsBRI1 invarious organs.

Total RNA (10 μg) from various organs of wild type plants were loadedinto each lane.

Organ specific expression of OsBRI1

(A) Leaf blade (lane 1), leaf sheath (lane 2), developed flower (lane3), rachis (lane 4), shoot apex (lane 5), root (lane 6), and seed (lane7).

Region Specific Expression of OsBRI1 in the Developing First Internode

(B) Node (lane 1), divisional zone (lane 2), elongation zone (lane 3,and elongated zone (lane 4) in developing internodes.

Differential Expression of OsBRI1 in Each Elongating Internode

(C) The divisional and elongation zones of the first to fourthinternodes, respectively, at the actively elongating stage for eachinternode (lanes 1–4). OsBRI1 was expressed at a high level also in theunelongated stem at the vegetative phase (lane 5).

Light-dependent and Brassinolide-dependent Expression of OsBRI1

(D) Rice seedlings were grown for ten days in the light (lanes 1 and 2)or dark (lane 3 and 4) on agar plate in the presence (lane 2 and 4) orabsence (lane 1 and 3) of 1 μM brassinolide.

FIG. 13 is a photograph which shows phenotype of the transgenic riceplants expressing the antisense strand of OsBRI1.

(A) Dwarf phenotype of OsBRI1 antisense plants with intermediate(centre) and severe phenotypes (right) compared to a wild type plant(left).

(B) Close up view of a transgenic plant with severe phenotype.

Bar=5 cm.

(C) Naked culm internodes of a transgenic plant. From left to right,wild type plant with normal elongation pattern of internodes andtransgenic plants with the dm, dm-d6, and d6 phenotypes are shown,respectively.

(D) Leaf morphology of wild type (left) and transgenic plants with mildphenotype (right), showing the erect leaves in the latter.

(E) Abnormal leaf morphology of a transgenic plant with a severephenotype, showing lack of developed sheath organs. Bar=10 cm.

(F) Panicle morphology in wild type (left) and transgenic plants withthe mild (centre) and intermediate (right) phenotypes

FIG. 14 is a photograph which shows the phenotype of a transgenic plantthat expresses dominant negative of OsBRI1. The transgenic plant (left)and a control plant containing vector without any inserts (right) areshown.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is specifically illustrated below with referenceto Examples, but it is not to be construed as being limited thereto.

Rice (Oriza sativa) seeds were soaked in distilled water and made toimbibe for 48 h at 30° C. After washing the seeds with distilled waterseveral times, the seeds were germinated in a dark chamber at 30° C. for8 days. Rice plants were grown in the field or in the greenhouse at 30°C. (day) and 24° C. (night).

EXAMPLE 1

Characterization of Rice d61 Dwarf Mutants

Rice dwarf mutants d61-1 and d61-2 were obtained by treatment withN-methyl-N-nitrosourea (NMU), respectively (FIG. 2A).

d61 mutants carrying the weak allele specifically fail to elongate thesecond internode (dm-type), while those with the strong allele fail toelongate all of the internodes except the uppermost one (d6-type) (Wu,X. et al. (1999) Bread. Sci. 49, 147–153).

The culm of d61-2 is much shorter than that of d61-1. In addition, d61-1shows typical dm-type pattern of internode elongation, while d61-2 showsthe d6-type (FIG. 2B). Thus, they were initially characterized as twoindependent mutants. However, crossing test demonstrated that they arealleles on a single locus. This is the first example of rice mutants ofa single locus which show different, specific patterns of inhibition ofinternode elongation.

These mutants have other abnormal phenotypes as a result of pleiotropiceffect, as well as that inhibiting internode elongation specifically.For example, the neck internode of the mutants is longer than that ofwild type plants (FIG. 2C). As the neck internode length shows aninverse relationship to the length of culm in these plants, wild typeplant thus has the longest culm and shortest neck internode, the d61-1mutant has intermediate length culm and neck internodes, and d61-2 hasthe shortest culm and the longest neck internode.

Another abnormal phenotype of these mutants is erect leaves (FIG. 2D).In wild type plants, the leaf blade bends away from the vertical axis ofthe leaf sheath towards the abaxial side. The leaf blade bends away fromthe leaf sheath at a specific organ, lamina joint, which is indicated byarrows in FIG. 2D. When the leaf blades and sheaths are fully elongated,cells at the adaxial side of the lamina joint start to elongate causingthe leaf blade to bend away form the leaf sheath. However, the leafblade of a mutant does not clearly bend away from the leaf sheath. Inthe d61-1 mutant some leaves still show slight bending (FIG. 2D,centre), but in the d61-2 mutant almost all the leaves are completelyerect (FIG. 2D, right).

That is, the degree of lamina inclination correlates to the severity ofthe dwarfism. The continuity of the severity in lamina inclinationsuggests that the longitudinal elongation of the surface cells on theadaxial side of lamina, which causes the lamina inclination, respondscontinuously to the brassinosteroid signal.

The lack of bending of the mutant leaves is not caused by none or lessdevelopment of the lamina joint. Indeed, even the d61-2 mutant with thesevere phenotype in lamina joint developed normally. The mutants showedshorter leaf sheaths than that of the wild type plants (FIG. 2E).

EXAMPLE 2

Observation of Cell Morphology in Internode

Internode elongation is caused by cell division in the intercalarymeristem and cell elongation in the elongation zone (Hoshikawa, K.(1989) Stem. In: The growing rice plant. (Nobunkyo), pp. 123–148).Therefore, dwarfing of the culms could be due to a defect in one or bothof these processes. To distinguish between these possibilities, thepresent inventors examined sections of each internode from adult plantsunder the microscope.

Developing or developed culms at various stages were fixed in FAA(formalin: glacial acetic acid: 70% ethanol, 1:1:18), and dehydrated ina graded ethanol series. The samples were embedded in a Technovit 7100resin (Kurzer, Germany) polymerized at 45° C. and 3–5 μm sections werecut, stained with Toluidine Blue and observed under the lightmicroscope.

FIG. 3 shows the cell morphology of the upper four internodes in a wildtype plant and the d61-2 mutant. In the wild type plant, cells in allinternodes were longitudinally elongated and well organized withlongitudinal files (FIGS. 3A, 3B, 3C, and 3D). Similar longitudinal cellfiles were also seen in the first internodes of the mutant plants,although the cells were a little shorter than those in the wild typeplant (FIG. 3E). In the non-elongated internodes of the mutant, such asthe second and third internodes, the arrangement of cells wasdisorganized with no organized cell files apparent (FIGS. 3F and 3G).Disorganization of the internodal cells indicates that the intercalarymeristems of the mutants, which normally give rise to the longitudinalfiles of elongated cells, are not developed in the non-elongatedinternodes. In the fourth internode, organized cell files were presentbut the cells were much shorter than that of the wild type plant(compare FIGS. 3D and 3H). This suggests that intercalary meristems diddevelop in these internodes but the cells failed to elongate.

EXAMPLE 3

Observation of the Arrangement of Microtubules

It was described in detail that cell elongation depends on theorientation of microfibrils (Ledbetter, M. C. and Porter, K. R. (1963)J. Cell Biol. 19, 239–250; Green, P. B. (1962) Science. 138, 1404).Therefore, the present inventors examined the arrangement ofmicrotubules in the internodal cells of wild type and d61-2 mutantplants by immunofluorescence microscopy.

Microtubules in internodal parenchyma tissue were strainedimmunofluorescently. More specifically, internodal parenchyma tissue wasprefixed for 45 min at room temperature in 3.7% (w/v) paraformaldehydein microtubule-stabilizing buffer (0.1 M piperazine-diethanolsulphonicacid, 1 mM MgCl₂, 5 mM ethyleneglycol-bis(3-aminomethylether-N,N,N′,N′-tetraacetic acid, 0.2% (v/v) Triton X-100,1% (w/v) glycerol, pH 6.9). Longitudinal sections were cut with a freshrazor blade, collected in the fixation solution, treated for 40 mintherein, and washed in the fixation solution without paraformaldehyde.The sections were then incubated with a rabbit anti-α-tubulin monoclonalantibody, diluted 1:500 in phosphate-buffered saline containing 0.1%(v/v) Triton X-100, for 1 h at 37° C. The sections were washed threetimes in the buffer without anti-serum and then incubated overnight at4° C. with fluorescein-isothiocyanate-labeled mouse anti-rabbit IgGantibody diluted 1:50 in phosphate-buffered saline containing 0.1% (v/v)Triton X-100. After three washes in the same buffer, they were mountedin antifading solution (Fluoro Guard Antifade reagent, Bio-Rad) andobserved under a fluorescence microscope.

As a result, in wild type plants, the microtubules in cells ofelongating internodes were arranged in an orderly manner at right anglesto the direction of elongation (FIG. 4A). However, in the d61-2 mutant,the microtubules in cells of the first elongating internode werearranged in different directions in each cell, apparently at random(FIG. 4B). In addition, the microtubules in the mutant appeared to bethinner and less distinct relative to those of the wild type plant.

Moreover, the present inventors were unable to observe any organizedmicrotubule arrangement in cells from non-elongated internodes of themutant plants.

Taken together with the results in Example 2 show that non-elongatedinternodes in the mutants fail to develop an intercalary meristem andthe cells lack an organized arrangement of microtubules. In thoseinternodes that do elongate in the mutants, although less than in thewild type plants, an intercalary meristem does develop but the cellslack a well-organized microtubule arrangement.

EXAMPLE 4

Test of the Sensitivity against Brassinosteroids

The dwarf phenotype and erect leaves of the d61 mutant suggests thepossibility that the D61 gene product could be involved in either thebiosynthesis or signal transduction of brassinosteroids. Thus, thepresent inventors carried out the following experiments.

First, the present inventors attempted to restore the dwarfism of thed61 mutants, but could not be achieved it by the application ofbrassinolide.

Next, seeds of the wild type and mutant plants were germinated on agarplates with or without 1 μM brassinolide. The characteristics of wholeseedlings were observed 1 day after germination.

The result showed that, when the wild-type plants were germinated on theplates with brassinolide, the coleoptiles of wild type plants elongatedabnormally resulting in a twisted shape and the foliage leaves grewpoorly and did not break through the coleoptile (FIG. 5). Furthermore,root elongation was inhibited and thus the roots were not straight butdeveloped in a wavy form. The wild type plants grew normally in theabsence of brassinolide, with coleoptile elongation stopping at an earlystage of germination and then foliage leaves elongating to break out ofthe coleoptile. Roots developed normally and did not have any wavy form(FIG. 5). In contrast to the wild type plants, the mutants showed normalgrowth patterns, as well as that of the wild type plants on plateswithout brassinosteroids, even in the presence of brassinosteroids.These results suggest that the mutant plants are less sensitive tobrassinolide than the wild type plants.

The present inventors further tested the sensitivity of the mutants tobrassinolide using a more quantitative method.

The degree of bending between the leaf blade and leaf sheath in rice iswell known to be sensitive to the concentration of brassinolide or itsrelated compounds. This unusual character of rice leaf is the basis fora quantitative bioassay for brassinosteroids, known as the lamina jointtest (Wada, Ketal. (1981) Plant and Cell Physiol. 22, 323–325), eventhough the biological function of endogenous brassinosteroids inmonocotyledonous plants including rice remains unknown. If the mutantsare less sensitive to brassinosteroids, the degree of bending betweenthe leaf blade and sheath of the mutant plants will be less than that ofthe wild type plants.

Lamina joints of first leave of wild type T65 plant (the backgroundstrain of the mutants), d61-1 mutant, and d61-2 mutant were tested with10⁻³ and 10⁻² μg/ml of brassinosteroids, respectively.

As a result, the leaf blade of wild type T65 plants was bent almost atright angles to the axis of the leaf sheath in the absence of exogenousbrassinolide becoming even more bent in the presence of increasingconcentrations of brassinolide (FIG. 6A). When the mutants were used forthe test, the degree of bending increased with higher concentrations ofbrassinolide as in the wild type plants. However, the absolute degree ofbending of the leaves from the mutants was much less than that of thewild type plants under the same conditions (FIGS. 6B and C). This wasparticularly evident with the d61-2 mutant in which control leaves werealmost straight and leaves treated with 10⁻² μg/ml brassinolide werebent at less than a right angle to the leaf sheath axis. The results ofthe lamina joint test confirm that the sensitivity of the mutants tobrassinosteroids is less than that of the wild type plants.

EXAMPLE 5

Quantitative Analysis of Brassinosteroids in the d61-2 Mutant

Example 4 demonstrates that the mutants have reduced sensitivity tobrassinosteroids. That is, mutants may synthesize higher concentrationsof brassinosteroids to compensate for the reduction. Thus, the presentinventors measured the concentration of brassinosteroids in both thed61-2 mutant and wild type plants using GC-SIM with internal standards.

Wild type and d61-2 plants were grown in a greenhouse with a 16-hrs dayand 8-hrs night. Shoots from 2-month old plants were harvested and thenimmediately lyophilized. Lyophilized shoots (50 g fresh weightequivalent) were extracted twice with 250 ml of MeOH—CHCl₃ (4:1 [v/v]),and deuterium-labeled internal standards (1 ng/g fresh weight) wereadded thereto. The extract was partitioned between CHCl₃ and H₂O afterevaporation of the solvent in vacuo. The CHCl₃-soluble fraction wassubjected to silica gel chromatography (Wako-gel C-300; Wako; 15 g). Thecolumn was sequentially eluted with 150 ml each of CHCl₃ containing 2%methanol and CHCl₃ containing 7% methanol. Each fraction was purified bySephadex LH-20 column chromatography, where the column volume was 200 mland the column was eluted with methanol-CHCl₃ (4:1 [v/v]). The fractionseluting from 0.6 to 0.8 (V_(e)/V_(t)) were collected as brassinosteroidfractions. After pre-purification on an ODS cartridge column (10×50 mm[internal diameter×column length]) in MeOH, the eluates derived from 7%MeOH fractions were subjected to ODS-HPLC at a flow rate of 8 ml/minwith 65% acetonitrile as the solvent. In HPLC purification, the 7%methanol eluate was resolved into castasterone (retention time from 10to 15 min), typhasterol (25 to 30 min), 6-deoxocastasterone (40 to 45min) fractions, and the 2% methanol eluate gave a 6-deoxotyphasterolfraction (55 to 60 min). Each fraction was derivatized and analyzed byGC-SIM. The endogenous levels of brassinosteroids were calculated fromthe ratio of the peak areas of the prominent ions from the endogenousbrassinosteroids and the internal standard.

As a result, brassinosteroid was not detected in shoots from either themutant or wild type plants, suggesting that brassinolide is a minorcomponent of the total brassinosteroids pool. However, all of the otherbrassinosteroids were detected in both plant types, with the exceptionof teasterone which was not found in the wild type plants. The contentsof all of the brassinosteroid compounds detected were greater in themutant plants (FIG. 7). In particular, castasterone was four timeshigher in the mutant than in the wild plant. These results support thehypothesis that the mutants have no sensitivity to brassinosteroids.

EXAMPLE 6

De-etiolation Phenotype of the d61 Mutants

Reduction of hypocotyl elongation and emergence of opening of thecotyledons and primary leaves in complete darkness are reported inArabidopsis mutants with deficiencies in brassinosteroid biosynthesis orbrassinosteroid signaling when grown in the dark (Kauschmann, A et al.(1996) Plant J. 9, 701–703; Szekeres, M. et al. (1996) Cell. 85,171–182).

This de-etiolated (DET) or constitutive photomorphogenesis (COP)phenotype in darkness is a common feature of Arabidopsisbrassinosteroids-related mutants. A similar DET or COP phenotype is alsoobserved in a tomato dwarf (d) mutant that shows a short hypocotyl, lackof apical hook, and expansion of cotyledons (Bishop, G. J. et al. (1996)Plant Cell. 8, 959–969). In contrast, a pea brassinosteroids-defectivemutant, 1 kb, does not show such a DET phenotype (Nomura, T. et al.(1997) Plant Physiol. 93, 572–577). Mutants were grown in the dark todetermine whether such DET or COP phenotypes were also found inmonocotyledonous plants and whether the d61 rice mutants showedcharacteristics of skotomorphogenesis.

As a result, when wild type plants were germinated in the dark, theyshowed unusual elongation of the mesocotyl and internodes compared tolight-grown seedlings (FIG. 8A). Such elongation of the mesocotyl andinternodes did not occur in the mutants even in the dark (FIGS. 8B and8C). This failure of the mesocotyl and internodes to elongate in thedark is not a common characteristic of rice dwarf mutants. For example,two other dwarf mutants, d18 and d35, which are deficient in gibberellinbiosynthesis, showed elongated mesocotyls and internodes when grown inthe dark (FIGS. 8D and 8E, respectively).

Thus, it is conceivable that the reduced elongation of the mesocotyl andinternodes are specific feature of the d61 mutants and that the d61mutants have de-etiolated phenotype. In addition, it is also indicatedthat de-etiolation due to defects in brassinosteroid signal is commoncharacteristic in both dicotyledonous and monocotyledonous plants. Thatis, it is conceivable that brassinosteroid signals are important forskotomorphogenesis in both dicotyledonous and monocotyledonous plants.

EXAMPLE 7

Mapping and Linkage Analysis of D61 Locus

For mapping of the D61 locus, the present inventors crossed the d61-2mutant with an Indica rice cultivar, Kasarath (Oriza sativa L. cv.Kasarath). The linkage analysis between the mutant phenotype andrestriction fragment polymorphism (RFLP) markers released from the RiceGenome Project (Tsukuba, Japan) revealed that the D61 locus maps to thelong arm of chromosome 1, with tight linkage to the RFLP marker, C1370(FIG. 9A).

As d61 could be characterized as a mutant with no or reduced sensitivityto brassinosteroid, the present inventors also tested the linkagebetween the mutant phenotype and a rice gene that is homologous to theArabidopsis BRI1 gene.

The Arabidopsis BRI1 gene was isolated as the only gene that is involvedin brassinosteroid signal transduction (Li, J. and Chory, J. (1997)Cell. 90, 929–938). Furthermore, the present inventors carried out aBLAST search to identify one rice EST clone, S1676, with high homologyto the Arabidopsis BRI1 gene (Li, J. and Chory, J. (1997) Cell. 90,929–938).

Rice genomic DNA was isolated from leaf tissue using an ISOPLANT DNAisolation kit (Nippon GENE Co., Japan). One μg of the genomic DNA wasdigested with appropriate restriction enzymes and transferred ontoHybond N⁺ membranes (Amersham) under alkaline conditions. The membranewas probed using the partial cDNA fragment (corresponding to the regionfrom Ser 740 to Asp 1116 in the kinase domain), which specificallyhybridized with the genomic DNA fragment encoding OsBRI1. All of thesteps were carried out according to the method described by Church andGilbert (1984) PNAS. 81, 1991–1995, except that membranes werehybridized at high stringency (68° C.).

An RFLP between the Japonica (12.5 kb) and Indica (17.5 kb) rice wasobserved when genomic DNAs were digested with EcoRI and probed with thecDNA clone. All F2 plants with the mutant phenotype were homozygous forthe Japonica allele (12.5 kb), whereas the F2 plants with the wild typephenotype were either homozygous for the Indica allele (17.5 kb) orheterozygous with both the Japonica and Indica alleles (12.5+17.5 kb,FIG. 9B). This result demonstrates that the D61 locus is closely linkedto the position of the rice gene that is homologous to the ArabidopsisBRI1.

EXAMPLE 8

Identification of the d61 Gene

The above linkage analysis strongly suggested that the d61 mutation iscaused by loss of function of the rice homologue of the Arabidopsis BRI1gene. To test this possibility, the present inventors screened ricegenomic DNA library with probes and isolated the entire length. of therice BRI1 homologous gene (OsBRI1, Oryza sativa BRI1). Hybridization inthis screening was performed as described in Church and Gilbert (1984)except that membranes were hybridized at higher stringency (68° C.).Sequencing was carried out according to the same method described byChurch and Gilbert.

The structure of the OsBRI1 gene is quite similar to the ArabidopsisBRI1 gene in its entire length (FIG. 10 and FIG. 11). The predictedOsBRI1 polypeptide contains several domains that are also present in theBRI1, and the functions of which were discussed (Li, J. and Chory, J.(1997) Cell. 90, 929–938). These domains consist of a putative signalpeptide, two conservatively spaced cysteine pairs, a leucine-rich repeatdomain, a transmembrane domain, and a kinase domain. The N-terminus ofthe predicted OsBRI1 polypeptide has a hydrophobic segment which ispredicted to act as a signal peptide to transport the protein to theplasma membrane. In the BRI1 protein, Li and Chory predicted a potential4-hepted amphipathic leucine zipper motif following the signal peptide(Li, J. and Chory, J. (1997) Cell. 90, 929–938), but OsBRI1 does nothave such a typical leucine zipper motif in the corresponding region.

A putative extracellular domain (from Met¹ to Leu⁶⁷⁰), consisting of 22tandem copies of a leucine-rich repeat (LRR) of about 24-amino acidswith 12 potential N-glycosylation sites (Asn-X-Ser/Thr), is flanked bypairs of conservatively spaced cysteines. The LRR has been implicated tofunction in protein—protein interactions (Kobe, B. and Deisenhofer, J.(1994) Trends Biochem. Science. 19, 415–421).

In comparison to the BRI1 sequence, OsBRI1 lacks three LRR domainscorresponding to the third to fifth repeat of the Arabidopsis BRI1. Thetwo LRRs before this deletion are less conserved except for theconsensus residues found between other LRR proteins, but the LRRs ofboth proteins are well conserved after the deletion in both the LRRconsensus residues and non-conservative amino acids. An unusual featureof the LRR region of BRI1 is the presence of a 70-amino acid islandbetween the 21st and 22nd LRR (Li, J. and Chory, J. (1997) Cell. 90,929–938). A highly similar feature is also present in OsBRI1 with thesame number of amino acids between the 18th and 19th LRRs correspondingto the site of island in BRI1. This unusual amino acid island in LRRregion must be important for functions thereof, because exchange of anamino acid residue in this island resulted in the loss of function ofBRI1 (Li, J. and Chory, J. (1997) Cell. 90, 929–938) This motif wasthought to be important for direct interaction with brassinosteroids orfor maintaining the structure of the brassinosteroids-binding domain(Li, J. and Chory, J. (1997) Cell. 90, 929–938).

The protein kinase domain of OsBRI1 has all eleven conserved subdomainsof eukaryotic protein kinases, retaining the invariant amino acidresidues in their proper positions (Hanks, S. K. and Quinn, A. M. (1991)Meth. Enzymol. 200, 38–62). The protein kinase domain of OsBRI1 ishighly related to that of BRI1 (44%) over the entire region. It is alsorelated to the kinase domains of other receptor-like protein kinases inhigher plants such as ERECTA (Torii, K. U. et al. (1996) Plant Cell. 8,735–746), CLV1 (Clark, S. E. et al. (1997) Cell. 3, 575–585), and RLK5(Walker, J. C. (1993) Plant J. 3, 451–456) from Arabidopsis, and Xa2lfrom rice (Song et al., 1995). The highly conserved structure of OsBRI1and these receptor-like protein kinases, especially in subdomains VIband VIII, suggests that OsBRI1 is a serine/threonine kinase rather thana tyrosine kinase (Hanks, S. K. and Quinn, A. M. (1991) Meth. Enzymol.200, 38–62).

EXAMPLE 9

Sequencing of OsBRI1 Gene in d61-1 and d61-2 Mutants

The present inventors also determined the entire sequences of the OsBRI1gene in the d61-1 and d61-2 mutants, and compared them to that of thewild type plant. The present inventors identified a single nucleotidesubstitution in each mutant allele at different sites (Table 1). Thegenomic mutation in d61-1 resulted in exchange from threonine toisoleucine at residue 989 in subdomain IX of the kinase domain which isconserved between OsBRI1 and BRI1. The genomic mutation in d61-2 changedvaline to methionine at residue 491 in the 17th LRR, just before theunusual 70-amino acid interrupting region. These mutations in the OsBRI1genes from the d61 mutants provide strong evidence that OsBRI1 encodesthe D61 locus.

TABLE 1 Alleles Characteristics of mutation Position of coding sequenced61-1 C → T Thr → Ile (989) d61-2 G → A Val → Met (491)

EXAMPLE 10

Molecular Complementation Analysis of the d61 Mutation by theIntroduction of OsBRI1 Gene

To confirm that OsBRI1 corresponds to the d61 locus, the presentinventors carried out complementation analysis of the d61-1 mutant byintroduction of the wild-type OsBRI1 gene.

More specifically, to confirm complementarity of d61 phenotype due tointroduction of a genomic OsBRI1 clone including its 5′ and 3′ flankingregions, a 10.5-kb restriction fragment including the entire codingregion was cloned into the XbaI-SmaI sites of the hygromycin resistancebinary vector pBI101-Hm3 (Sato, Y. et al. (1999) EMBO J. 18, 992–1002).pBI-cont was used as a control vector. The present inventors performedrice tissue culture and Agrobacterium tumefaciens mediatedtransformation.

Transformation of d61 with a control vector that carries no rice genomicDNA had no apparent effect on the culm length or the structure ofleaves. However, when a 10.5 kb DNA fragment containing the entirewild-type OsBRI1 gene was introduced, the normal phenotype was recoveredin almost plants that were resistant to hygromycin. This result confirmsthat the d61 mutant phenotype is caused by the loss-of-function mutationin the OsBRI1 gene.

EXAMPLE 11

RNA Hybridization Analysis of OsBRI1

Nothing is known about the function of endogenous brassinosteroids inmonocotyledonous plants. Therefore, the present inventors tested theexpression pattern of the OsBRI1 gene in various rice organs by RNAhybridization analysis.

RNA was isolated from various rice tissues as described in literature(Chomczynski, P. and Sacchi, N.: Anal. Biochem. (1987) 162:156). Ten μgof total RNA were electrophoresed in a 1% agarose gel, then transferredto a Hybond N⁺ membrane (Amersham), and analyzed by RNA gel blothybridization. The present inventors used a partial cDNA fragment(Ser⁷⁴⁰ to Asp¹¹¹⁶ corresponding to the kinase domain), as a probe whichspecifically hybridized to the genomic DNA fragment encoding OsBRI1. DNAhybridization analysis was performed with the same probe andhybridization conditions as described above.

As a result, a single, strongly-hybridizing band was detected in RNAfrom vegetative shoot apices (FIG. 12A). The size of the band wasapproximately 3.5 kb, which is almost the same size as the longest cDNAclone. More weakly-hybridizing bands of the same size were also observedin RNA from flowers, rachis, roots, and expanded leaf sheaths, while noor very faint bands were observed in RNA from expanded leaf blades.Thus, the expression of OsBRI1 varies markedly between organs suggestingthat the sensitivity to brassinosteroids also differs among theseorgans.

The present inventors also examined the expression of OsBRI1 inelongating culms (FIG. 12B). Elongating culms were divided into fourparts: the node and the division, elongation, and elongated zones of theinternode. As a result, the most strongly hybridizing band was found inRNA from the division zone. RNA from the elongation zone also gave astrong signal. RNA from the node gave only a weak signal, whilst thatfrom the elongated internode gave no signal at all. These resultsindicate that the elongating culm has different sensitivities tobrassinosteroids partially, with the most sensitive parts being thedivision and elongation zones where cells are actively dividing andelongating.

The present inventors further examined the expression of OsBRI1 in theelongation zones of the upper four internodes at the stage when eachinternode was actively elongating. A strongly hybridizing band was foundin RNA from the elongation zone of the uppermost (first) and the lowest(fourth) internodes, while relatively weak bands were seen with thesecond and third internodes (FIG. 12C). This result indicates that theinternodes differ in their sensitivity to brassinosteroids, with thesecond and third internodes being the least sensitive.

It was observed that the internodes differ in their sensitivity tobrassinosteroids. It suggests that the uppermost and fourth internodeshave higher sensitivity to brassinosteroids than the second and thirdinternodes, if the amount of OsBRI1 is a limiting factor inbrassinosteroid signal transduction. This idea is supported by themutant allele with the intermediate, dm-d6 type phenotype. These plantsshow specific reduction of the second and third internodes while theuppermost and fourth internodes are elongated. This is consistent withthe second and third internodes, with lower expression level of OsBRI1,having lower sensitivity to brassinosteroids such that they are unableto respond to the brassinosteroid signal and elongate. Presumably, thehigher OsBRI1 expression level in the uppermost and fourth internodesdoes allow these internodes to respond to the brassinosteroid signal andelongate. The higher expression level of OsBRI1 in the uppermost andfourth internodes can explain the unusual internode elongation patternof the dm-d6 type, but it cannot explain the occurrence of the d6 or dmtype. The d6 type, in which all of the internodes except the uppermostare reduced, could indicate that the uppermost internode is exposed tohigher levels of brassinosteroids than the fourth internode. The timingof the elongation of the uppermost internode corresponds with thedevelopment of anthers in the flowers, and high level ofbrassinosteroids have been observed in these organs in many plants(Grove, M. et al. (1979) Nature, 281, 216–217; Plattner, D. et al.(1986) J. Natural Products. 49, 540–545; Ikekawa, N. et al. (1988) Chem.Pharm. Bull. 36, 405–407; Takatuto, S. et al. (1989b) Agric. Biol. Chem.53, 2177–2180; Suzuki, Y. et al. (1986) Agric. Biol. Chem. 50,3133–3138; Gamoh, K. et al. (1990) Anal. Chim. Acta. 228, 101–105). Itappears that high levels of brassinosteroids move down from the anthersto lower organs, such as, the uppermost internode and induce internodeelongation and that the fourth internode completes its elongation beforeflower development and does not receive the high level brassinosteroidsignal from the flowers at the time of its active elongation. Thebrassinosteroid level and the sensitivity to brassinosteroids of OsBRI1cannot explain the specific retardation of the second internode observedin the dm-type mutants. Therefore, some other factor(s) must beinvolved. It seems likely that elongation of the second internode isregulated by several factors, since there are several independent dwarfmutants with the dm-phenotype including d1, d2, d11, and d61.

Very recently, the D1 gene was isolated and found to encode a proteinwith a similar structure to the α subunit (G-α) of a G protein(Fujisawa, Y. et al. (1999) Proc. Natl. Acad. Sci. USA. 96, 7575–7580;Ashikari, M. et al. (1999) Proc. Natl. Acad. Sci. USA. 96, 10284–10289)The D1 G-α like protein is now thought to be involved in the gibberellin(GA) signal transduction pathway, since the d1 mutant alleles show lowor no sensitivity to active GA. It is interesting that theloss-of-function mutants of the brassinosteroid signal-related protein,OsBRI1, and that of the GA signal-related protein, Gα, show the samephenotype, i.e., specific retardation of the second internode. Thus, inthe induction of elongation in the second internode, there could be aspecific mechanism common to brassinosteroids and GA signal transductionin the second internode.

Interestingly, high level expression of OsBRI1 was also seen in the stemat the vegetative stage, in which the internodes do not elongate,showing that high level expression of OsBRI1 in the culm does notnecessarily coincide with internode elongation.

EXAMPLE 12

Effect of Exogenous Brassinolide and Light on the Level of OsBRI1 mRNA

The present inventors also tested the effects of exogenously appliedbrassinolide and light on the level of OsBRI1 mRNA. Germinating seedswere placed on 0.9% agar plates with or without 1 μM brassinolide andgrown for six days in the light or dark.

As a result, on plates without brassinosteroids, the expression level ofOsBRI1 in dark grown seedlings was higher than in light grown seedlings(FIG. 12D).

This suggests that the dark-grown rice seedlings have a highersensitivity to brassinosteroids than the light-grown plants (Worley, J.F. and Mitchell, J. W. (1971) J. Amer. Soc. Hort. Sci. 96, 270–273).High sensitivity to brassinosteroids in dark-grown plants will due tothe elongation of internode cells in situations where the cells inlight-grown plants do not respond to brassinosteroids.

Furthermore, on plates with brassinosteroids, both of the light- and thedark-grown rice seedlings had a reduced level of OsBRI1 expression.

In contrast to rice, the level of BRI1 expression in Arabidopsis islittle changed between dark- and light-grown seedlings (Li, J. andChory, J. (1997) Cell. 90, 929–938). The reason for this differencebetween the expression patterns of the rice OsBRI1 and that of theArabidopsis BRI1 is not known. However, the difference could be relatedto the difference in photoresponse mechanisms that rice is short-dayplant, while Arabidopsis is long-day plant.

EXAMPLE 13

Phenotypic Analysis of Transgenic Plants Expressing the Antisense Strandof OsBRI1

The above phenotypic analyses of the d61 mutants and the singlenucleotide exchange in the OsBRI1 genes in each mutant suggest that theymight not be null alleles and could retain some partial function. Toinvestigate further the function of brassinosteroids in rice, thepresent inventors attempted to generate other mutants with more severephenotypes by overexpression of the antisense strand of the OsBRI1transcript under the control of the rice Actin1 gene promoter (Zang, W.et al. (1991) Plant Cell. 3, 1155–1165).

For constructing Actin1 promoter::antisense OsBRI1, apromoter-terminator cassette (pBIAct1nos) containing the Act1 promoter(Zhang, W. et al. (1991) Plant Cell. 3, 1155–1165) and NOS terminatorwas constructed by substitution of the Act1 promoter for the 35Spromoter in the hygromycin resistance binary vector, pBI35Snos (Sato, Y.et al. (1999) EMBO J. 18, 992–1002), which contains the 35S promoter andNOS terminator, between the HindIII and XbaI sites. The cDNA cloneencoding entire OsBRI1 coding region was introduced between the XbaI andSmaI sites of pBIAct1nos. Vector pBI-cont, containing no insert was usedas a control vector. For reduction of OsBRI1 expression, theOsBRI1-antisense cDNA were introduced into the rice cultivar Nipponbare.The present inventors performed rice tissue culture and Agrobacteriumtumefaciens mediated transformation.

Almost all of the resulting transgenic plants (90% or more, 18 out of20) produced erect leaves during the early stages of seedling growth(FIG. 13D). All of the transformants (20 out of 20) showed a dwarfedphenotype of varying severity (FIG. 13A). In the plants with the weakestphenotype, the length of each internode was partially and uniformlyreduced resulting in an elongation pattern similar to that of the wildplant (FIG. 13C) (dn-type mutants). Plants with intermediate phenotypeshad the typical internode elongation patterns of dm-type (specificreduction of the second internode, FIG. 13C) or d6-type (specificreduction of the second to fourth internodes, FIG. 13C) mutants or amixed dm- and d6-type phenotype (specific reduction of the second andthird internodes, FIG. 13C). Plants with the severe phenotype onlyformed abnormal leaves without developed sheath organs and theinternodes did not elongate (FIG. 13E). These kinds of plants were lessthan 15-cm high, even after cultivated for one year or more, and did notproduce seeds (FIG. 13B). The other phenotypes were inherited insubsequent generations and cosegregated with hygromycin resistance. Thecosegregation between the abnormal phenotypes and hygromycin resistance,and the similarity between the intermediate phenotype of the antisenseplants and the d61 mutants demonstrate that the antisense strand acts tosuppress the function of OsBRI1 in the transgenic plants.

EXAMPLE 14

Phenotype of Transgenic Plants Expressing Dominant Negative of OsBRI1

Transgenic rice containing the kinase region of the OsBRI1 gene undercontrol of the rice actin 1 gene promoter (Zhang, W. et al., (1991)Plant Cell, 3: 1155–1165) was produced to analyze the function of therice brassinosteroid receptor. That is, the plasmid was constructed asfollows so that only the carboxy terminal kinase domain ofbrassinosteroid receptor would be expressed without the amino terminusregion from the first methionine to 738th glycine. The present inventorsused a pair of the primer,5′-GGCTCTAGACAGCCATGGCGAGCAAGCGGCGGAGGCTG-3′/SEQ ID NO: 4 (5′-primer:which includes TCTAGA as XhoI site, CAGCC added to increase translationefficiency, ATG as the-initiation codon, an additional GCG encodingalanine, and AGC encoding 739th serine, following further nucleotidesencoding amino acids after 740th residue of the wild type sequence,lysine, arginine, arginine, and leucine) and5′-AGATCTACTCCTATAGGTA-3′/SEQ ID NO: 5 (3′-primer: which includes AGATCTas XbaI site and following 3′-untranslated region). These two primerswere used to amplify the kinase region of the brassinosteroid receptor.The amplified fragment was then digested with XbaI and inserted into apBI vector between XbaI-SmaI sites. The vector pBI-cont which does notcontain the insert was used as a control.

The rice cultivar Nipponbare was used to produce transgenic plants whichexpresses kinase domain to control OsBRI1 expression. Rice tissueculture and Agrobacterium tumefaciens mediated transformation wereperformed.

As a result, most of the transgenic plants (90% or more: 25/27individuals) formed erect leaves at an early stage of seeding growth(FIG. 14). The length between the internodes was partially and equallyshortened. The phenotype was inherited to the progeny by co-segregationwith hygromycin resistance activity. The co-segregation between theabnormal phenotype and hygromycin resistance and similarities betweenthe dominant negative plants and the d61 mutant intermediate phenotypeindicate that the partial cDNA for the kinase portion has activity inthe transgenic plant and suppresses OsBRI1 function.

INDUSTRIAL APPLICABILITY

The present invention provides a gene and a protein which functions toincrease rice brassinosteroid sensitivity. This gene is involved inelongation of plant internode cells and inclination of leaves.Therefore, it is possible to produce phenotypically modified plants bycontrolling this gene. For example, by suppressing the expression of thegene of the present invention, dwarf plants, which are resistant tolodging and which enables planting a higher number of individuals perunit area, can be produced, which is significant in the production ofcrop products. It is also possible to produce ornamental plants havingnew aesthetic value by dwarfism of height or culm length of said plantsvia suppression of expression of DNA of the present invention. On theother hand, brassinosteroid sensitivity of the plant can be increased byintroducing and expressing the DNA of the present invention in plants,resulting in increase of height of the plant and yield of the wholeplant. This is useful especially in increasing yield of plants foranimal feed.

1. An isolated DNA being selected from the group consisting of: (a) DNAencoding a protein comprising the amino acid sequence of SEQ ID NO: 2;and (b) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or
 3. 2.The DNA of claim 1, wherein the DNA is cDNA or isolated genomic DNA. 3.The DNA of claim 1, wherein the DNA is derived from a monocotyledonousplant.
 4. The DNA of claim 3, wherein the DNA is derived from a plant ofthe Gramineae family.
 5. A vector which comprises the DNA of claim
 1. 6.A transformed cell which comprises the DNA of claim
 1. 7. A transformedplant, or a progeny, clone, or breeding material thereof, comprising theDNA of claim
 1. 8. The isolated DNA of claim 1, wherein the DNA encodesa protein consisting of the amino acid sequence of SEQ ID NO:2.
 9. Theisolated DNA of claim 1, wherein due DNA comprises SEQ ID NO:1 or
 3. 10.The Isolated DNA of claim 1, wherein the DNA consists SEQ ID NO:1 or 3.